It has been over two decades since it was demonstrated using histochemical methods that plaques and tangles, the pathological hallmarks of Alzheimer's disease (AD), contain activity specific to cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The same methodology demonstrated that these AD-related cholinesterases (AD-ChE) possess different properties when compared with cholinesterases found in normal neuronal cell bodies and axons, are likely to originate in glial cells and may possess unusual activities. Biochemical studies have indicated that ChEs may be involved in the pathology of AD, particularly as it relates to the genesis of plaques and production of their main constituent, the amyloid- (A) peptide. It has been suggested that in addition to current ChE inhibitor therapies aimed at ameliorating the cortical cholinergic deficit in AD, it would also be desirable and perhaps more beneficial to inhibit ChEs in plaques and tangles, due to their possible participation in the disease process. However, research regarding the ChEs in plaques and tangles is currently at a stand-still because the only methods to date that have allowed distinction between AD-ChEs and normal ChEs are based on histochemical demonstration of activity in tissue sections. There is no method available to specifically isolate ChEs from plaques and tangles. Therefore, there is no direct information on the biochemical properties of AD-ChEs and whether they make a specific contribution to disease pathology. Furthermore, the development and pre-clinical testing of ChE inhibitors that can specifically target AD-ChEs requires a method which can isolate these enzymes from tissue. Taking advantage of recent observations at our laboratory, the fact that some ChEs are anchored to tissues (such as muscle) with the aide of extracellular matrix proteins heparan sulphate proteoglycans (HSPG) and that HSPG are also present in plaques and tangles, the experiments in the present application will test two novel methods for isolation of ChEs from plaques, tangles and glia. The specific aims of the proposed research will test the following hypotheses: 1. Cholinesterases from plaques, tangles and glial cells but not neurons and axons in fixed and fresh frozen tissue sections will be passively released into storage buffer over time. 2. Cholinesterases from plaques, tangles and glial cells but not neurons and axons in fixed and fresh frozen tissue will be released upon treatment with various glycosaminoglycans, heparinases or proteases. 3. Released cholinesterases will display molecular and enzymatic characteristics and inhibition patterns identical to those demonstrated histochemically in plaques, tangles and glia and biochemically in AD brain. The successful isolation and characterization of AD-ChEs will allow mechanistic studies of the possible detrimental effects of these ChE species; will aide in design and testing of new inhibitors specifically targeting AD-ChEs; and, is likely to lead to investigation of AD-ChEs as biomarkers of AD in blood and CSF. Plaques and tangles of Alzheimer's disease contain the cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with altered properties that have been suggested to participate in the disease process. However, currently there are no methods available to isolate AChE and BuChE specifically from plaques and tangles, and the present application aims to fill in this gap. The successful isolation and characterization of AChE and BuChE from plaques and tangles will allow mechanistic studies of the possible detrimental effects of these enzyme species, will aide in design and testing of new inhibitors specifically targeting cholinesterases in plaques and tangles, and is likely to lead to investigation of plaque and tangle cholinesterases as biomarkers of AD in blood and cerebrospinal fluid. [unreadable] [unreadable] [unreadable]